新生兔气管软骨细胞的生物学特性

Biological characteristics of newborn rabbit tracheal chondrocytes

目的:探讨体外分离培养的新生兔气管软骨细胞的生物学特性。方法:通过酶消化法体外分离培养新生兔气管软骨细胞;倒置显微镜观察软骨细胞形态及生长状况;电镜观察软骨细胞超微结构;运用real-time PCR、免疫细胞化学染色和甲苯胺蓝染色检测气管软骨细胞分泌的细胞外基质成分。结果:体外分离、培养的兔气管软骨细胞呈短小三角形或不规则形贴壁生长。超微结构显示细胞较多突起,孔隙较多,胞质丰富,细胞器发达,细胞内可见大量蛋白分泌物。软骨细胞表达I、II型胶原、蛋白聚糖等,以II型胶原和蛋白聚糖表达为主。免疫细胞化学染色II型胶原和SOX9阳性,I型胶原弱阳性。甲苯胺蓝染色阳性。结论:适宜的酶消化单层培养法获得的新生兔气管软骨细胞具有分泌软骨细胞外基质成分的特性,可初步为体外构建组织工程气管治疗新生兔气管狭窄的实验研究提供种子细胞。

AIM： To investigate the biological characteristics of newborn rabbit tracheal chondrocytes in vitro. METHODS ： Newborn rabbit tracheal chondrocytes were obtained by the method of enzyme digestion, and then cultured in monolayer in vitro. Morphological and growth observations were performed under inverted phase contrast microscope. The ultrastructures of the cells were observed under scanning electron microscope and transmission electron microscope. The bi- ological characteristics of secreted extracellular matrix components were detected by real-time PCR, immunocytochemistry staining and toluidine blue staining. RESULTS： Newborn rabbit tracheal chondrocytes isolated and cultured in vitro showed short triangular or irregular shapes, and adherent growth very well. The ultrastrnctures of the ceils showed pore and abundant cytoplasm and organelles, with a lot of protein secretions in the cells. The chondrocytes expressed the mRNA of collagen I, collagen II and proteoglycans, mainly collagen II and proteoglycans. Immunocytochemistry staining showed col- lagen II and SOX9 positive, and eoUagen I weakly positive. To[uidine blue staining was also positive. CONCLUSION： Enzyme digestion and monolayer culture are suitable method to obtain newborn rabbit tracheal chondrocytes. These cells, secreting extracellular matrix components, are able to be selected as seed cells for tissue engineering of trachea in vitro, and used to study the therapeutic method for neonatal rabbit tracheal stenosis.